(1) Field
The general field of our present invention is the art of quantitative measurement of analytes such as immunogens or haptens dissolved in biological fluids.
(2) Prior Art
Competitive protein-binding assays and radioimmunoassays are used for measurements of substances in biological fluids which require that the substance be labeled with a radioisotope in order to achieve the desired specificity and sensitivity.
Other assay methods use an enzyme label to obviate problems of radioactivity labeling. One type of enzyme labeling method uses a competitive immunoassay between unlabeled antigen and the antigen labeled with an enzyme for an antibody bound to an immunosorbent; Engvall and Perlmann were among the first to disclose this technique in their paper entitled Enzyme-Linked Immunosorbent Assay, ELISA, 8 Immunochemistry 871 (1971), and others have subsequently published variations of their general approach. A second type of enzyme labeling method employs antibody (rather than antigen) labeled with enzyme and antigen covalently bound to an immunosorbent; antigen in the unknown sample is assayed in a non-competitive reaction with the enzyme labeled antibody, followed by separation of antigen/labeled antibody complexes. Masseyeff and Maiolini have co-authored articles on this technique published in 19 Biomedicine 314 (1973), 6 J. Immuno. Methods 355 (1975) and 8 J. Immuno. Methods 223 (1975). A third type of enzyme immunoassay as reported by Rubenstein et al in 47 Biochem. Biophys. Res. Commun. 846 (1972), see also U.S. Pat. No. 3,877,837 to Rubenstein et al, utilizes enzyme covalently linked to the analyte. In the case of hapten assay, antibody to the hapten will bind the hapten/enzyme conjugate and sterically inhibit enzymatic activity. The hapten to be assayed by this technique will compete for antibody with the hapten/enzyme conjugate, thereby decreasing the inhibition of enzyme activity, and the subsequent increase in enzymatic activity will be proportional to the original amount of hapten present.
Prior art methods discussed briefly above are described in greater detail in Yorde et al, Competitive Enzyme-Linked Immunoassay etc., 22 Clinical Chemistry 1372 (1976), which paper is hereby incorporated by reference as to its discussion of the prior art and our present invention. Voller et al review various immunoassays in their article Enzyme Immunoassays in Diagnostic Medicine, 53 Bull. World Health Organ. 55 (1976).
Our new immunoassay for soluble immunogens and haptens is believed to offer advantages over the prior methods referred to above, as discussed in detail in the ensuing description.